In the prior art there is described an extraction method (Journal of Natural Products 53, 1249-55, 1990), which reported that paclitaxel content in the extract in the range of between 0.009% and 0.07%. After removing the hexanes soluble impurities (waxes, pigments, etc.) and after partition between water and dichloromethane, the paclitaxel concentration in the dichloromethane soluble crude extract was in the range of between 0.03% and 0.3% by weight. Paclitaxel (Taxol) is represented by the following structural formula: ##STR1##
is an anticancer compound. Paclitaxel exhibits a unique mechanism for preventing the growth of cancer cells by affecting the microtubules, which plays an important role in cell division and other cellular functions.
Paclitaxel is clinically effective for the treatment of retractory human ovarian and breast cancer and has exhibited promising activity against a number of other types of cancers such as liver, peritoneal, cervical, prostate, colon, and esophageal cancers.
Paclitaxel was primarily extracted from the bark of pacific yew Taxus brevifolia. Unfortunately, the yew grows very slowly, approximately eight inches per year and thus the bark is a limited source of paclitaxel. This lead researchers to seek alternative means for producing paclitaxel and analogues thereof which may display superior antitumor activity, such as derivatives of 9-dihydrotaxol that have enhanced water solubility. As an example 13-acetyl-9-dihydrobaccatin III, a taxane diterpene, has been found to be a useful precursor to make such analogues. This has been discussed in U.S. Pat. No. 5,440,056, issued to Klein et al., Aug. 8, 1995.
13-Acetyl-9-dihydrobaccatin III and baccatin III are represented by the following structure formulas: ##STR2##
This can be obtained from, for example, Taxus canadensis which is more widely distributed than Taxus brevifolia. Hence, 13-acetyl-9-dihydrobaccatin III is available more abundantly than paclitaxel.
Conventional methods for the isolation of taxanes, including paclitaxel, 13-acetyl-9-dihydrobaccatin III, and baccatin III generally involve the steps of extracting taxanes from a biomass with an alcoholic solvent and separating and purifying the individual taxane by chromatography.
Prior art methods disclose the use of various types of chromatographic technologies to separate paclitaxel and derivatives thereof. For example, U.S. Pat. No. 5,380,916, issued to Rao, Jan. 10, 1995, describes a process using C18 reverse phase liquid chromatography. Although reverse phase chromatography can be successful in separating the taxanes, this protocol employs an expensive absorbent and requires a significant time investment. Accordingly, this process is not economically and industrially viable.
U.S. Pat. No. 5,478,736, issued to Nair, Dec. 26, 1995, discloses a process using a silica gel normal phase absorption chromatography to purify taxanes. In normal phase absorption chromatography, Al.sub.2 O.sub.3 or silica gel is used as an absorbent which is about 100 times less expensive than the ordinary reverse phase absorbent. The problem for the normal phase silica gel absorption chromatography is that the absorbent will bind with paclitaxel which will not be eluted by solvent mixture, therefore the product yield is reduced.
U.S. Pat. No. 5,530,020, issued to Gunarvardana et al., Jun. 25, 1996, disclosed a process for isolating 13-acetyl-9-dihydrobaccatin III from Taxus canadensis which employs planet coil countercurrent chromatography (PCCC). Some of the disadvantages associated with this procedure are that it is complex, consumes significant amounts of solvent and can be used to purify only a small amount (milligrams) of samples.
So far, no distributive column chromatography for separating and purifying paclitaxel and related taxanes has been disclosed.